| Methods for detection of mycobacterium tuberculosis -> Monitor Keywords |
|
|
  Methods for detection of mycobacterium tuberculosisUSPTO Application #: 20070072188Title: Methods for detection of mycobacterium tuberculosis Abstract: The present invention provides an oligonucleotide primer pair having SEQ ID NO: 3 and SEQ ID NO: 4 for amplification of Early Secretory Antigenic Target (esat)-6-gene of Mycobacterium species. The invention also provides a method for detecting M .tuberculosis in a sample based on the amplification of esat-6 gene, comprising isolating DNA template from the sample, amplifying with the above oligonucleotide primer pair and subjecting the amplified DNA product to separation and staining to detect the presence of amplified DNA product for identifying Mycobacterium tuberculosis in the sample. The invention further provides a diagnostic kit for detection of Mycobacterium tuberculosis. The invention also provides a method of detecting Mycobacterium tuberculosis from a sample by amplifying the 16s rRNA region from the isolated DNA template by conventional methods to detect Mycobacterium species and further amplifying the positive sample contains Mycobacterium species using primers positive for ESAT-6 region detection of Mycobacterium tuberculosis. (end of abstract) Agent: Birch Stewart Kolasch & Birch - Falls Church, VA, US Inventors: Sarman Singh, Pawan Sharma USPTO Applicaton #: 20070072188 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070072188. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD [0001] The invention provides novel oligonucleotide primers for the amplification of Early Secretory Antigenic Target (ESAT)-6 regions for detection of Mycobacterium species. The primers are used for differentiating the Mycobacterium tuberculosis from other species of Mycobacterium. Further, the invention provides a method for detection of Mycobacterium tuberculosis based on the DNA amplification of the ESAT-6 region. BACKGROUND AND PRIOR ART [0002] According to the WHO, tuberculosis still kills 3 million individuals per year, making it the leading infectious cause of death. It is believed that one in every three individuals on the planet harbor the causative microorganism belonging to the genus Mycobacterium (13, 24). This genus represents a complex phenotypic and genotypic diversity amongst its more than 100 odd species (4,9,10,14,15,17,27,28,30,34). Though, the most important human pathogenic species is Mycobacterium tuberculosis (M.tb), other species commonly known as non- tubercular Mycobacteria (NTM) or Mycobacteria other than tuberculosis (MOTT) also cause human infections in various clinical forms, particularly in immunosuppressed patients, adding to the human suffering in terms of morbidity and mortality. The cases of HIV-TB co-infections are rapidly increasing, after the AIDS epidemic, from both the developed and developing world (2, 10, 13, 18-21, 24). Most of these non-Tubercular Mycobacteria are not susceptible to the conventional anti-tubercular treatment and are wrongly considered due to infection with multi-drug resistant (MDR) strains of M. tuberculosis (5-7, 10, 21, 26, 31, 32). Many of these MDR labeled infections are actually not caused by the drug resistant strains of M. tuberculosis but by the non-tubercular Mycobacteria. HIV-Mycobacteria co-infection is the most common killer opportunistic infection in Indian AIDS patients (31), but the outbreaks of infections due to the NTM in these severely immunocompromised population, can not be ruled out (19). Hence it is very important to identify the causative agent to the species level for appropriate treatment. [0003] However, in India the diagnosis of Mycobacterial infections is established empirically on clinico-radiological basis or only by sputum smear examination, and the infections caused by non-tubercular Mycobacteria are under-diagnosed due to lack of diagnostic facilities. The speciation of Mycobacteria using conventional methods is very slow, labor intensive, hazardous and not always reproducible (33) and hence left unattempted by most of the Indian Laboratories. [0004] To overcome the shortcomings of conventional methods of species specific identification of Mycobacteria, the molecular techniques are being more commonly used, in the recent years. Molecular identification methods are rapid, highly sensitive and specific and can be used on a large number of samples (1, 6, 9, 16, 33, U.S. Pat. No. 5,652,106, U.S. Pat. No.5,731,150). One molecular target which has proved to be the most promising and commonly used is the 16S rRNA gene analysis. This gene is conserved in all the species of Mycobacterial genus (1, 15, 16, U.S. Pat. No. 5,811,269). Once the genus mycobacterium is identified, the species can be differentiated by southern hybridization using the species specific probes, or gene sequencing (4, 11, 22, 28). However, identification of all the non-tubercular species of Mycobacteria may require several specific primers and repeated experimentation and most often it may not be necessary in a resource poor setting, particularly from the fresh cases, when several species can easily be identified on the basis of phenotypic characters. Therefore, the present invention intends to differentiate the Mycobacteria tuberculosis from non-tubercular Mycobacterium species, using a rapid PCR amplification method. For this, a novel, simple and rapid method for differentiating MTB and NTM has been developed, that uses a pair of oligonucleotide primers targeting the esat-6 and 16S rRNA genes. [0005] The 16S rRNA gene amplification method is a good tool to identify all the Mycobacteria. However, its further utility is restricted only for taxonomic purposes after sequencing the amplified gene product. Therefore, the single PCR method can not answer the most important clinical question, whether the Mycobacterium is M. tuberculosis or other than M. tuberculosis. Therefore, with clinical point of view, it is extremely important that the Clinical Microbiologist not only diagnoses a Mycobacterial infection in his patient but also provides the identification of the etiological agent, whether it is M. tuberculosis or other species of Mycobacterium for which the treatment protocol is totally different from the First. On the other hand, the novel oligonucleotide primer pair designed in the present invention target the esat-6 gene which is Mycobacterium tuberculosis specific gene coding for the early antigen of 6 kDa mass (3,8, 23,25). Though this antigen has been used in the humoral immunodiagnosis as well as for evaluating the cell mediated immune response against M. tuberculosis, replacing PPD with this antigen in skin testing (3, 8). More recently, it has also been used for vaccination against tuberculosis (23, 25, U.S. Pat. No. 6,649,170) but its gene has never been used as a target for molecular diagnosis of tuberculosis. This gene is deleted in species of Mycobacteria other than tuberculosis complex (4). [0006] The antigen- antibody based test methods have been a research topic but no antigen has been found to be satisfactory. Most of the genus-specific antigens failed because they cross reacted with the environmental Mycobacteria and with BCG which is given as a vaccine. Even the M. tuberculosis specific antigen (ESAT-6 antigen in this case) has a fundamental drawback of poor predictive value to make an organ specific disease diagnosis. The detection of antibodies against ESAT-6 protein has been found to be somewhat useful in tuberculosis non-endemic countries, its utility in TB endemic countries such as whole of Asia, Africa, Russia and other South American countries is very limited due to sub-clinical exposures of the population . Obviously all the exposed persons will have circulating antibodies in their blood against this antigen. Therefore, if a pre-exposed asymptomatic person (antibodies already positive) gets fresh TB brain infection (TB meningitis) and in another pre-exposed person there is no such fresh infection, the antibody detection assays will not be useful for the specific diagnostic use in such cases, as both these patients will be positive for these antibodies. To explain it in other words, antibody detection assays, for diseases of high endemicity (e.g. Tuberculosis which is airborne) have very poor specificity and organ specific diagnosis can not be made at all, as the antibody detection methods are indirect evidences of infection. [0007] On the other hand the molecular methods such as PCR are highly specific because in these methods the genome of the living organism from the specific diseased site is detected. In other words, using PCR amplification, the specific diagnosis of tubercular meningitis, abdominal tuberculosis, gastrointestinal tuberculosis, genitourinary tuberculosis besides the pulmonary tuberculosis can be made. Also the PCR amplification will detect only active diseases and not the old exposures. Moreover the biggest advantage of molecular methods is that the DNA of causative agent (Mycobacterium) can be detected from old samples such as mummies, fossils etc. while the antibodies can not. [0008] The present invention shows its utility in species-specific and rapid molecular diagnosis of tuberculosis using esat-6 gene amplification, for the first time. Definitions of Certain Terms Used in the Specification [0009] AIDS--Acquired Immunodeficiency Syndrome; [0010] M.tb--Mycobacterium tuberculosis, [0011] NTM--Non-tubercular Mycobacteria; [0012] MDR-TB--Multi drug resistant tuberculosis BRIEF DESCRIPTION OF ACCOMPANYING DRAWING [0013] FIG. 1 The PCR method for genus-specific identification of the Mycobacterium species based on the 16s rRNA gene. Lanes 1, 11, & 22 are 100 bp molecular weight markers. Lanes 2, 12, & 23 are H37rv standard strain of Mycobacterium tuberculosis. Lanes 3-10 are standard non-tubercular Mycobacteria (as shown in table 1), 13-21 and 24-26 are clinical isolates. [0014] FIG. 2: The isolates which were identified as Mycobacteria on the basis of genus-specific 16s rRNA PCR were subjected to amplification based on the ESAT-6 region. Lane 1 & 17 is 100 bp molecular weight markers. Lane 2 & 18 is H37rv standard strain of Mycobacterium tuberculosis. Lane 3-15 & 19-24 is the same clinical isolates as shown in panel A. The lane 16 is M. bovis showing no amplification. OBJECT OF THE INVENTION [0015] The main object of the present invention is to develop an oligonucleotide primer pair for specific amplification of the Early Secretory Antigen of Target (esat)-6 gene of Mycobacterium species [0016] Another object of the present invention is to develop a method for detecting M. tuberculosis in a sample based on the amplification of esat-6 gene using the primer pair. [0017] Still another object of the present invention is to develop a method for detecting M. tuberculosis wherein the amplification is done by polymerase chain reaction. [0018] Further object of the present invention is to develop a diagnostic kit for detecting M. tuberculosis, based on the amplification of (esat)-6 gene. SUMMARY OF THE INVENTION Continue reading... Full patent description for Methods for detection of mycobacterium tuberculosis Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods for detection of mycobacterium tuberculosis patent application. Patent Applications in related categories: 20080108057 - Allelic imbalance in the diagnosis and prognosis of cancer - Methods for assessing the extent of allelic imbalance in a genomic nucleic acid sample. Methods for diagnosing cancer and determining the prognosis of a patient with cancer, including breast or prostate cancer, by assessing the extent of allelic imbalance in a genomic nucleic acid sample. ... 20080108069 - Forensic identification - The invention provides allelic ladder mixtures and individual alleles suitable for use in such mixtures. The allelic ladder mixtures give improved identification and distinguishing capabilities, particularly suitable in forensic investigations. ... 20080108079 - Genes associated with copd - A method of screening a small molecule compound for use in treating COPD, comprising screening a test compound against a target selected from the group consisting of the gene products encoded by CELSR3, CHRNA5-THRU-CHRNB4, GPR55, LGR8, PMPCB, SENP1, UCHL1, UQCRC1, BRD2, CCK, HTR6, KCNK3, MBTPS2, NCOA6, PRSS7, SMO, THRA, or ... 20080108078 - Genes associated with migraine - A method of screening a small molecule compound for use in treating Migraine, comprising screening a test compound against a target selected from the group consisting of the gene products encoded by APOE, GNAL, NEDD4L, PDIP, TPCN1, TRPM8, ADRA1B, P2RX4, TAAR2, TAAR3, USP11, CHRNA5, RAB5A, DPP8, F2RL1, FZD5, PTGER1, SPI, ... 20080108080 - Genes associated with obesity - A method of screening a small molecule compound for use in treating obesity, comprising screening a test compound against a target selected from the group consisting of the gene products encoded by IRS1, IL12A, ADAMTS7, APG4C, CITED1, GGTLA1, PKD1, TSC2, APG4B, CST7, CXCL5, GPR75, CAPN9, DPYS, F13A1, HFE, GPR173, A2M, ... 20080108077 - Genes associated with rheumatoid arthritis - A method of screening a small molecule compound for use in treating rheumatoid arthritis, comprising screening a test compound against a target selected from the group consisting of the gene products encoded by ACHE, ADAMTS16, AGER, BAT3, BRD2, C2, BF, C4A-THRU-TNXB, C6ORF21, LY6G6D, CACNA1D, CCR4, CLIC1, DNM1, EDG1, FAS, HLA-DQB1, ... 20080108076 - Genes associated with unipolar depression - A method of screening a small molecule compound for use in treating unipolar depression, comprising screening a test compound against a target selected from the group consisting of the gene products encoded by ADCYAP1R1, HMGB1, MIP, NIPSNAP3A, SRC, WFS1, CLIC6, GABRR3, KDR, PKD1L1, ADARB2, MAP3K1, PPARGC1A, DRD3, PTHR1, BF, CART, ... 20080108081 - Genetic polymorphisms associated with coronary stenosis, methods of detection and uses thereof - The present invention is based on the discovery of genetic polymorphisms that are associated with coronary stenosis. In particular, the present invention relates to nucleic acid molecules containing the polymorphisms, variant proteins encoded by such nucleic acid molecules, reagents for detecting the polymorphic nucleic acid molecules and proteins, and methods ... 20080108075 - Kits and methods for assessing oxidative stress - The invention relates to kits and methods for assessing the susceptibility of a human to oxidative stress or damage. The methods involve assessing occurrence in the human's genome of one or more polymorphisms (e.g., single nucleotide polymorphisms) that occur in one or more genes associated with oxidative stress and that ... 20080108072 - Maize event dp-098140-6 and compositions and methods for the identification and/or detection thereof - Compositions and methods related to transgenic glyphosate/ALS inhibitor-tolerant maize plants are provided. Specifically, the present invention provides maize plants having a DP-098140-6 event which imparts tolerance to glyphosate and at least one ALS-inhibiting herbicide. The maize plant harboring the DP-098140-6 event at the recited chromosomal location comprises genomic/transgene junctions having ... 20080108074 - Methods and compositions for efficient nucleic acid sequencing - Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the ... 20080108071 - Methods and systems to determine fetal sex and detect fetal abnormalities - Non-invasive methods for determining the sex of a human fetus and predicting other genetic abnormalities are disclosed. The methods include screening a maternal sample for biomarkers known to be associated with risk of genetic abnormalities; removing all or substantially all nucleated and anucleated cell populations from the maternal sample to ... 20080108073 - Methods of analysis of methylation - Methods for determining the methylation status of a plurality of cytosines are disclosed. In some aspects genomic DNA target sequences containing CpGs are targeted for analysis by multiplex amplification using target specific probes that can be specifically degraded prior to amplification. The targets may be modified with bisulfite prior to ... 20080108070 - Methods, compositions, and kits for the detection and monitoring of colon cancer - Methods and compositions for the diagnosis and monitoring of colon cancer are disclosed. ... 20080108082 - Polymerase enzymes and reagents for enhanced nucleic acid sequencing - Compositions that include DNA polymerases having increased residence times for nucleotide analogues, particularly modified recombinant Φ29-type DNA polymerases with such increased residence times, are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA amplification are also provided. Compositions including α-thiophosphate nucleotide analogues with four ... ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Methods for detection of mycobacterium tuberculosis or other areas of interest. ### Previous Patent Application: Methods for amplification of nucleic acid sequences using staggered ligation Next Patent Application: Methods for drug discovery, disease treatment, and diagnosis using metabolomics Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Methods for detection of mycobacterium tuberculosis patent info. IP-related news and info Results in 1.64164 seconds Other interesting Feshpatents.com categories: Daimler Chrysler , DirecTV , Exxonmobil Chemical Company , Goodyear , Intel , Kyocera Wireless , |
||
